PROJECT SUMMARY The overall objective of the PNNL Proteome Characterization Center (PCC) is to integrate genomic information with proteomic technologies to comprehensively characterize the proteome and post-translational modifications (PTMs) as well as the functional interaction of proteins in signaling networks in cancers. As one of the PCCs in the current Clinical Proteomic Tumor Analysis Consortium (CPTAC) program, PNNL successfully applied advanced proteomics capabilities and expertise to the comprehensive proteogenomic and phosphoproteomic characterization of ovarian high-grade serous carcinomas from The Cancer Genome Atlas, and has developed novel mass spectrometric approaches to facilitate the analysis of intact proteins, low stoichiometry PTMs, and extremely small samples. The planned PNNL PCC will build on these achievements and contribute to the success of the CPTAC network by characterizing multiple new tumor types assigned by CPTAC, using both the proven standardized, multiplexed discovery and targeted confirmatory workflows as well as introducing novel technologies and platforms, as appropriate, to improve proteomic measurements. Both human biospecimens and preclinical models provided by NCI will be analyzed. The Discovery Research Arm will comprehensively characterize the proteome and multiple PTM-omes in human biospecimens and preclinical samples of 2-3 cancers with the validated advanced proteomics platforms, integrated workflows and informatics tools, using highly multiplexed isobaric labeling and state-of- the-art liquid chromatography-tandem mass spectrometry instrumentation at a throughput of up to 300 samples per year. The Confirmatory Research Arm will verify the impact of genomic variation on the changes in proteome and PTM-omes with validated targeted proteomics platforms (e.g., multiple reaction monitoring), peptide standards and quality assurance. Up to 100 highly specific, multiplexed targeted proteomics assays for both proteins and PTMs will be developed each year for the confirmatory studies using up to 150 samples per year. We will also refine and validate several developmental strategies and platforms at the PNNL PCC leveraging our on-going technology development in areas such as ultrafast gas phase separations and small- sized sample analysis platforms and methodologies to further improve the throughput and sensitivity in both the discovery and confirmatory studies. The PNNL PCC will work closely with the other PCCs, Proteogenomic Data Analysis Centers and Proteogenomic Translational Research Centers in the CPTAC network on data integration and bioinformatics analysis, as well as translational applications.